The goal of the proposed research is to investigate arachidonic acid metabolites (eicosanoids) in the physiology of tick salivary glands and their role as salivary secretory products that facilitate tick feeding. Eicosanoids are secreted by the salivary glands of several tick species and are hypothesized to assist in tick feeding by preventing host hemostatic reactions and increase the flow of host blood at the tick feeding site. Preliminary data indicate that high concentrations of arachidonic acid are present in phosphatidylcholine and phosphatidylethanolamine in the salivary glands of A. americanum. Because nothing is known about the sequestration and/or synthesis of arachidonic acid in the salivary glands or changes in levels in the salivary glands during tick feeding in any tick, these questions will be studied in A. americanum. In most tissues, the synthesis of eicosanoids is limited by the availability of their common precursor, free arachidonic acid which is liberated from membrane phospholipids. We propose to establish the organelle locations of arachidonic acid and how the free level of arachidonic acid is liberated from esterified stores in complex lipids of the salivary glands. Activity and control of phospholipase A2 will be studied in detail. We will investigate agonist induced increased in phospholipase A2 activity and its linkage to GTP- binding proteins and modulation by calcium, protein kinase C and lipocortin-like molecules. The ability of the salivary glands to metabolize arachidonic acid both in vivo and in vitro will be studied. These experiments will be performed in Nebraska with salivary gland extracts shipped from Oklahoma. Eicosanoids will be separated, identified, and quantified by TLC, HPLC, RIA and enzyme-linked immunoassay (EIA). Saliva from A. americanum will be collected by stimulating ticks to secrete with known agonists of secretion and analyzed for eicosanoids and pharmacological activity involving smooth muscle bioassays and platelet aggregation. These experiments will be performed in Arizona with tick saliva collected in Oklahoma. We will also study the possibility that eicosanoids serve as "local" hormones to control or modulate secretion. Results of these experiments will enable us to have a clearer picture of the importance of arachidonic acid metabolites at the tick-host interface and the importance of these molecules in enabling ticks to be efficient vectors of pathogens.